The science and instrumentation of spectroscopy as developed over the last century has become increasingly expanded and specialized as the various methods and applications of analysis came into existence. Today, spectroscopy has been divided into individual and distinctly different methods and instrumentation systems for: ultraviolet and visible spectrophotometry; fluorescence and phosphorescence spectrophotometry; flame emission and atomic absorption spectrometry; atomic emission spectroscopy; infrared spectrophotometry; raman spectroscopy; nuclear magnetic resonance spectroscopy; electron spin resonance spectroscopy; refractometry and interferometry; and various others. Of these, the optical sensors and optical sensing detection systems utilizing the ultraviolet and visible absorption methods and the fluorescence and phosphorescence excitation and emission systems are perhaps the best known and commonly utilized.
The essentials of an ultraviolet/visible spectrometry instrumentation system utilize the principles of absorption photometry; and comprise, in its simplest forms, a light energy source, focusing optics, an unknown or standard sample cuvette, a wavelength isolation device, and a detector with amplifier and readout system. From an engineering standpoint, it is desirable that this type of absorption photometry system be detector limited - that is, the limiting factor should be the noise generated by the detector. Anything that can be done to increase signal levels at the detector is therefore desirable. The measure of performance is usually defined as precision, or photometric accuracy. In terms of construction, one recognizes the differences between single-beam and double-beam light paths; and whether the photometer module is a direct reading or employs a balance circuit. Other available instrumentation features include double monochromatation and dual wavelength systems.
In comparison, fluorescence and phosphorescence is a physical phenomenon based upon the ability of some molecules to absorb and then emit light. With these molecules, the absorption of light energy (photons) at specified wavelengths is followed by the emission of light from the molecule of a longer wavelength and at a lower energy state. Such emissions are called fluorescence if the emission lifetime is relatively short-lived, typically a rate of from 10.sup.-9 to 10.sup.-7 seconds. Phosphorescence lifetimes usually are longer and fall within the range from 10.sup.-4 to 10 seconds. The most striking difference between the two emission forms are the conditions under which each type of photoluminescence is observed. Fluorescence is usually seen at moderate temperature in the liquid solution. Phosphorescence is seen in rigid media, usually at very low temperatures.
A simple generalized instrument suitable for fluorescence and phosphorescence spectrophotometry usually comprises a source of light energy; a primary filter or excitation monochromator; a sample cell; a secondary filter or emission monochromator; a photodetector; and a data readout device. In contrast to ultraviolet/visible instrumentation, two optical systems are necessary. The primary filter or excitation monochromator selects specific bands or wavelengths of radiation from the light source and directs them through the sample in the sample cell. The resultant emission or luminescence is isolated by the secondary filter or emission monochromator and directed to the photodetector which measures the intensity of the emitted radiation. For observance of phosphorescence, a repetitive shutter mechanism is required.
For more complete and detailed information, the following publications and references are provided, the text of which are expressly incorporated by reference herein: Willard, Merritt, Dean, and Settle, Instrumentation Methods Of Analysis, 6th edition, Wadsworth Publishing Company, Belmont, Calif., 1981; Joseph R. Kakowicz, Principles Of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Skoog and West, Fundamentals Of Analytical Chemistry, 4th edition, Saunders College Publishing, 1982.
A more recent event has been the development of fiber optic sensors and instrumentation systems utilizing ultraviolet, visible, and/or fluorometric photometry techniques. Such fiber optic sensors and sensing apparatus are fast becoming established analytical tools for remote and in-situ optical sensing determinations. The development of fiber optic sensors and their applications are illustrated by the following publications: Angel, S. M., Spectroscopy 2:38-48 (1987); Hilliard, L. A., Analytical Proceedings 22:210-224 (1985); Boisde et al., Talanta 35:75-82 (1988); Wolfbeis, O. S., Pure & Appl. Chem. 59:663-672 (1987); Seitz, W. R., Anal. Chem. 56:16A-34A (1984); and Seitz, W. R., CRC Critical Reviews In Analytical Chemistry 19:135-173 (1988).
Regardless of the light energy system and photometric basis employed, an ideal optical sensor must have the ability to measure the concentration of an analyte continuously over the entire range of changes in the optical properties of the sensing reagent. To date, this sensor ability has been based on the availability of suitable, long-lasting, reversible chemistries and reagents. The systems are thus based and dependent upon the ability of the reagent to first associate and then disassociate reversibly with the specific analyte--a requirement which has eliminated many colorimetric and fluorometric compositions and reactive ligands from being used in such sensors because these compositions are irreversible in their reactions. Accordingly, because many ultraviolet, visible, phosphorescent, and fluorescent compositions form a tightly binding complex with the analyte of interest or utilize reagents which generate an irreversibly colored or fluorescent adduct product for reaction with the analyte of interest, these compositions and photometric techniques have been generally avoided and deemed inappropriate for use with fiber optic sensors [see for example: U.S. Pat. No. 4,666,672; Vo-Dinh et al., Appl. Spectros. 41:235-738 (1987); Sutherland et al., Clin. Chem. 30:1533-1538 ( 1984); and Tromberg et al., Anal. Chem. 59:1226-1230 (1987)].
In those limited numbers of optic probes utilizing irreversible chemistries, these probes may be employed if they operate in an integrating mode; however, they must be replenished frequently with fresh sensing reagent ligands because of the irreversible nature of their reaction with the analyte to be detected Clearly, therefore, a fiber optic sensor which releases irreversible reagents and ligands reactive with an analyte of interest and which does not require frequent replenishment of reagents and provides accurate and reliable modes of delivery would be recognized and appreciated by ordinary practitioners within this art as a major improvement and substantive advance in this field.